The alphaviruses comprise a group of 26 animal viruses many of which cause serious illness in man or in domestic animals. The applicants wish to understand the origin, evolution, and spread of these viruses in nature and to understand the molecular biology of their replication and how this relates to the virulence of the virus and the ability to cause disease. In the coming project period, the applicants propose research in two areas. (1) The applicants have hypothesized that alphaviruses originated in the Americas some thousands of years ago and were spread to the Old World in two waves to form the Sindbis group and the Semliki Forest group. To provide support for this hypothesis, the applicants propose to study Mayaro virus, a member of the Semliki Forest group present in the Americas, and to study a number of viruses belonging to the Sindbis group present in both the Americas and in the Old World. These studies will attempt to trace the spread of the alphaviruses throughout the world and to determine the time that this occurred. These studies are also important for the eventual control of alphavirus infections, which annually cause very many cases of serious human illness. (2) The applicants will examine the functions of the virus-encoded proteins in RNA replication. The applicants have developed a model for RNA replication that proposes that polyproteins are essential components of the alphavirus RNA replicase early in the infection cycle. Cleavage of these polyproteins by the virus nonstructural proteinase converts the early replicase to a late replicase, which results in the shutoff of minus strand RNA synthesis and the efficient synthesis of genomic and subgenomic plus strand RNAs. Concomitant with this, the infected cell becomes resistant to superinfection by closely related viruses. The applicants propose a number of molecular genetic experiments and experiments using a two hybrid system to further develop this model and to examine the interactions of the virus-encoded nonstructural proteins with one another and with cellular proteins. The applicants also propose to examine the contributions of the virus structural proteins to RNA replication.